The development of impaired kidney function is a common complication of human sepsis that doubles the risk for mortality. The pathogenic mechanisms that underlie renal tubule dysfunction during sepsis are poorly understood and the identification of new and effective therapeutic approaches remains a priority. Studies in our laboratory combining methods for study of microdissected renal tubules with a mouse cecal ligation and puncture (CLP) sepsis model uncovered new mechanisms through which sepsis impairs the transport function of the medullary thick ascending limb (MTAL). Sepsis reduces the ability of the MTAL to absorb HCO3- through a novel two-hit mechanism involving a decrease in intrinsic HCO3- absorptive capacity and enhanced inhibition by lipopolysaccharide (LPS) through Toll-like receptor 4 (TLR4). Preliminary studies indicate that these mechanisms play a direct role in the pathogenesis of sepsis-induced metabolic acidosis, a risk factor for increased morbidity and mortality in septic patients. However, the specific receptor signaling and transport mechanisms that are targeted by sepsis to impair MTAL function remain poorly defined. In addition, we have made the novel observation that MTAL HCO3- absorption is inhibited directly by the endogenous damage- associated molecule high mobility group box 1 (HMGB1), identifying a new pathophysiological mechanism for renal tubule dysfunction during sepsis. Further studies demonstrate that treatment with the nontoxic TLR4- based immunomodulator monophosphoryl lipid A (MPLA) prevents sepsis-induced inhibition of MTAL HCO3- absorption and reduces the severity of acidosis in CLP mice. Accordingly, the Specific Aims are: AIM I: Test the hypotheses: 1) that the NHE1 and NHE3 Na+/H+ exchangers are targets for impaired function during sepsis, whereby CLP decreases basal HCO3- absorptive capacity of the MTAL through inhibition of NHE1 and enhances inhibition of HCO3- absorption by LPS through TLR4-mediated inhibition of NHE3, and 2) these inhibitory effects are mediated through ERK signaling and play a role in sepsis-induced acidosis. We will also test the hypothesis that TLR2 plays a critical and previously unrecognized role in mediating sepsis-induced renal tubule dysfunction. AIM II: Test the hypothesis that HMGB1 decreases MTAL HCO3- absorption through a receptor for advanced glycation end products (RAGE)-Rho kinase 1 signaling pathway and determine the significance of this pathway for sepsis-induced MTAL dysfunction and acidosis. AIM III: Test the hypothesis that MPLA stimulates the PI3K-Akt pathway through TLR4 and Trif, which prevents sepsis-induced inhibition of HCO3- absorption through downregulation of ERK. These studies will use a multidisciplinary approach to provide new information on signaling and transport mechanisms through which TLR4, TLR2, and HMGB1- RAGE inhibit HCO3- absorption in the MTAL, to identify a novel role for these pathways in the impairment of MTAL HCO3- absorption and the pathogenesis of metabolic acidosis in sepsis, and to determine molecular mechanisms by which the therapeutic agent MPLA prevents sepsis-induced MTAL dysfunction.